We have recently reported an important role of c-Jun N-terminal protein kinase (JNK) in apoptosis caused by various substrates of ethanol-inducible cytochrome P450 2E1 (CYP2E1) such as acetaminophen (APAP), 4-hydroxynonenal (HNE), carbon tetrachloride, and long chain fatty acids as well as a non-CYP2E1 substrate such as troglitazone, which causes hepatic damage. As a part of our continued studies on the apoptosis mechanism, we studied the mechanism of cell death by ethanol, because the early signaling mechanism of cell or organ damage by ethanol is poorly understood. Our results show that ethanol caused time- and dose-dependent cell death in SK-N-SH neuroblastoma cells and HCT116 human colon cancer cell used as models in our study. Ethanol increased the activities of JNK and p38 kinase in a time- and concentration dependent manner. However, activation of both JNK and p38 kinase seemed important in ethanol-induced cell death, because pretreatment with a respective inhibitor of JNK or p38 kinase significantly reduced the activity of each kinase and the rate of ethanol-induced apoptosis. We also observed that ethanol increases the level of caspase 8-dependent Bid cleavage, a critical factor for promoting mitochondrial apoptosis. This event was significantly blocked by pretreatment of cells with a selective inhibitor of caspase 8. These results strongly indicate that ethanol promotes apoptosis by activating the JNK and p38 kinase as well as promoting Bid cleavage. Heavy alcohol consumption negatively affects the functions of various cells and tissues. The detrimental effects of alcohol mainly result from: changes in redox state through alcohol metabolism, production of reactive aldehydes, generation of reactive oxygen and nitrogen species, reduction in protective anti-oxidants including mitochondrial glutathione, elevation of tumor necrosis factor alpha and other cytotoxic cytokines, changes in the activities of mitogen activated protein kinases, etc. These alcohol-mediated events lead to a state of oxidative stress and damage. However, it is still unknown which proteins are sensitive to oxidative modification and their roles in oxidative damage after alcohol exposure. Therefore, we sought to identify oxidized proteins and their potential role in ethanol-mediated oxidative damage. E47 HepG2 hepatoma cells with transduced CYP2E1 were known to undergo apoptosis upon exposure to various toxic substrates of CYP2E1 including ethanol and APAP. Mitochondrial fractions from E47 hepatoma cells, treated with 100 mM ethanol for different times, were treated with biotin-conjugated iodoacetate to detect oxidized cysteine residues in all proteins. Protein-bound biotin was subsequently detected by streptavidin-HRP followed by enhanced chemiluminescence. Our results showed that 70, 54, and 48 kDa proteins were modified at 4 and 8 h, long before the actual cell death observed at 16 or 24 h after ethanol exposure. Modification of these proteins also took place after exposure to 25, 50 and 100 mM ethanol for 8 h. Because of the time differences in protein modification (observed at early times) and apoptosis (at later times) after alcohol exposure, we believe that these oxidized proteins may be critically important in cellular protection against ethanol-mediated oxidative injury. Because of the similar molecular size and active site cysteine (Cys302), we speculate that the 54 kDa mitochondrial protein may be aldehyde dehydrogenase 2 (ALDH2). To confirm our hypothesis, the identity of each of the oxidized proteins and potential changes in their functions after ethanol exposure are being determined. We are also analyzing the mitochondrial proteins from ethanol-treated mouse liver to correlate with the in vitro results. This approach would provide new information about the protein targets of oxidative modification and their role in oxidative damage.